利用SPAG8分离精子与上皮细胞混合体系中精子的试验研究李学博12.doc

利用SPAG8分离精子与上皮细胞混合体系中精子的实验研究 李学博1,2，王清山2，宁淑华3，王业全2，封宇2，缪元颖2，李红卫2（250014 济南，山东省高校证据鉴识重点实验室（山东政法学院）1；400021重庆，重庆市公安局物证鉴定中心2；400020重庆，重庆市公安局江北分局3） [摘要] 目的 利用精子特异性抗体SPAG8（Sperm associated antigen 8）结合免疫磁珠技术分离混合细胞系中的精子，建立混合斑中分离检验精子细胞DNA的新方法。方法 制备含有精子103、104及105个左右/mL及阴道上皮细胞104个左右/mL的3中混合悬液及模拟混合斑各10例，分别以生物素标记的SPAG8单克隆抗体孵育特异结合混合细胞系中的精子，然后用亲和素包被的免疫磁珠通过生物素-亲和素复合体定向捕获并分离精子细胞，提取分离后的精子DNA，进行STR复合扩增和DNA分型检测。结果 免疫磁珠-亲和素-生物素-抗体复合物可分离混合细胞体系中的精子，当混合悬液内的精子数量达到103个/mL时，90%的样本可得到13个STR基因座的单一男性分型，精子数量达到104个/mL时，90%的样本均可得到16个STR基因座的单一男性分型；含有104个左右/mL以上数量精子的混悬液制作的混合斑，单一男性13个STR基因座的正确分型率达80%以上。结论 该方法简便快捷，适用于新鲜混合斑检材的检验，可作为混合斑法医学检验的有效补充手段。 [关键词] SPAG8；免疫磁珠；混合斑；精子；DNA [中图法分类号] R89；R392.3 Application of sperm-specific antibodies SPAG8 for the separation of sperm from sperm and epithelial cell mixtures Li Xuebo1,2, Wang Qingshan2, Ning Shuhua3, Wang Yequan2, Feng Yu2, Miao Yuanying2, Li Hongwei2 [1 Key Laboratory of Evidence Identification in Colleges and Universities of Shandong Province (Shandong University of Political Science and Law), Jinan 250014; 2Institute of Forensic science, Chongqing Public Security Bureau, Chongqing 400021; 3Jiangbei Branch of Chongqing Public Security Bureau, Chongqing 400021] [Abstract] Objective To establish a new method for the separation of sperm from sperm and epithelial cell mixtures by apply sperm-specific antibodies SPAG8 (Sperm associated antigen 8) and immunemagnetic beads. Method Three different concentration mixed cell suspensions and stain of sperm and vaginal epithelial cell was prepared which containing vaginal epithelial cells approximately 10 000/mL and sperms approximately 1 000/mL, 10 000/mL and 100 000/mL, respectively. Biotin-labeled SPAG8 monoclonal antibody and avidin-coated immunomagnetic beads was incubated with the mixed cell systems successively and the sperm cells were connected with the magnetic beads by the antibody-biotin-avidin system. The DNA of sperm cells was extracted and amplified using Identifiler? plus kit PCR reagents. PCR